Abstract

The present work was aimed at studying the ability of three model green proteins to covalently bind to microparticles (MPs) based on poly(D,L-lactic acid) (PLA). Green fluorescent protein (sfGFP), the fusion protein of recombinant human β2-microglobulin (β2M) with sfGFP (β2M–sfGFP) and the fusion protein of recombinant human amylin (IAPP) with sfGFP (IAPP–sfGFP) were isolated using affinity chromatography. MP–PLAs were formed by the double-emulsion method. The modification of MP–PLAs by protein was confirmed by laser scanning microscopy (LSM). In addition, LSM was used to study the phagocytosis of MP–PLA modified by different proteins and free model proteins by macrophages. Recombinant sfGFP was shown to binds to the surface of particles at lower amounts compared to β2M–sfGFP and IAPP–sfGFP. This is probably due to the fact that protein amino groups that could potentially react with activated carboxyl groups on the surface of particles are sterically inaccessible for this reaction because of the sfGFP structure. The β2M and IAPP proteins, being components of the respective recombinant fusion proteins, are spacer structures between the surface of spherical particles and sfGFP. It was established that a threefold increase in the protein/particles ratio did not lead to an increase in the bound protein per unit of particle mass, which may indicate the amount of protein that can be bound per unit of particle mass is limited by the capacity of particles themselves. The study of phagocytosis of protein-modified MP–PLAs has shown that MP–PLAs containing model proteins (β2M–sfGFP and IAPP–sfGFP) on their surface are successfully phagocytized by macrophages and, thereby, can contribute to the activation of cell-mediated immune response, which is important for controlling various, including viral, infections. Phagocytosis of model proteins (β2M–sfGFP, IAPP–sfGFP) has also been shown in the present work. This may be due to the fact that both β2M and IAPP are amyloidogenic and aggregation-prone proteins. In all likelihood, the aggregates of these proteins can be absorbed by macrophages due to the increased size compared to their monomeric forms.

中文翻译：

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免疫细胞对蛋白质修饰聚合物微粒的吞噬作用

摘要

目前的工作旨在研究三种模型绿色蛋白与基于聚（D,L-乳酸）（PLA）的微粒（MP）共价结合的能力。使用亲和层析分离绿色荧光蛋白（sfGFP）、重组人β2-微球蛋白（β2M）与sfGFP的融合蛋白（β2M-sfGFP）以及重组人胰淀素（IAPP）与sfGFP的融合蛋白（IAPP-sfGFP）。MP-PLA 采用双乳液法形成。激光扫描显微镜 (LSM) 证实了蛋白质对 MP-PLAs 的修饰。此外，LSM还用于研究巨噬细胞对不同蛋白修饰的MP-PLA和游离模型蛋白的吞噬作用。与 β2M–sfGFP 和 IAPP–sfGFP 相比，重组 sfGFP 与颗粒表面的结合量较低。这可能是由于以下事实：由于 sfGFP 结构，可能与颗粒表面上的活化羧基反应的蛋白质氨基在空间上无法接近。β2M和IAPP蛋白是各自重组融合蛋白的组成部分，是球形颗粒表面和sfGFP之间的间隔结构。已确定蛋白质/颗粒比率增加三倍不会导致每单位颗粒质量的结合蛋白增加，这可能表明每单位颗粒质量可结合的蛋白质量受到容量的限制粒子本身。对蛋白质修饰的 MP-PLAs 吞噬作用的研究表明，表面含有模型蛋白（β2M-sfGFP 和 IAPP-sfGFP）的 MP-PLAs 可以成功地被巨噬细胞吞噬，从而有助于激活细胞介导的免疫反应，这对于控制各种感染（包括病毒感染）非常重要。目前的工作还显示了模型蛋白（β2M–sfGFP、IAPP–sfGFP）的吞噬作用。这可能是由于 β2M 和 IAPP 都是淀粉样蛋白和易于聚集的蛋白。与单体形式相比，这些蛋白质的聚集体很可能由于尺寸增加而被巨噬细胞吸收。